Hypertension is a major risk factor for heart attack, stroke, and kidney failure and contributes to over 350,000 deaths annually in the United States. Although hypertension is thought to be one of the most modifiable contributors to cardiovascular disease, our incomplete knowledge of the etiology of this complex disease trait has hindered our ability to therapeutically control blood pressure in a diverse population of affected patients. Recent studies in pre-clinical models indicate that the small GTPase protein, RhoA, regulates blood pressure by stimulating the contractility of smooth muscle cells (SMC) within resistance vessels. Although studies from our lab and others have shown that RhoA activity is regulated by cellular proteins that control its on/off state, the precise signaling mechanisms that control its activity in blood vessels are largely unknown. We have recently demonstrated that the Rho-specific GTPase activating protein, GRAF3, is highly and selectively expressed in SMC in mice and humans and inhibits RhoA-dependent contractility in this cell-type. Moreover, our data indicate that GRAF3's ability to limit RhoA activity in SMC is required for the maintenance of normal blood pressure and provide a novel mechanism for the blood pressure (BP) associated locus within the GRAF3 gene that was recently identified by Genome Wide Association Studies (GWAS). Interestingly, the BP- associated locus maps to the 80Kb first intron of the GRAF3 gene strongly suggesting that its effects on BP are mediated by alterations in GRAF3 expression. Indeed, the minor GRAF3 allele that decreases blood pressure is significantly associated with increased GRAF3 mRNA levels in human tibial artery samples. Taking advantage of our considerable expertise on the regulation of SMC-specific gene expression, we have already identified regulatory elements within the hypertensive locus that exhibit SMC-selective transcriptional activity and have shown that a minor allele variation within one of these elements significantly increased its transcriptional activity. The goal of this multi-PI, multidisciplinary proposal is to further examine the role of GRAF3 in the pathogenesis of human hypertension. In aim 1 we will use our unique GRAF3-deficient mouse lines in combination with several well-characterized hypertensive models to determine the extent to which changes in vessel tone contribute to the development of hypertension and hypertension-induced cardiovascular disease. The goal of Aim2 is to identify the regulatory elements and transcription factors that drive SMC-specific GRAF3 expression and to identify the SNPs that alter the activities of GRAF3 regulatory elements. The goal of Aim3 is to test whether GRAF3 genotype correlates with GRAF3 levels and hypertension in patient populations. To this end, we have established collaborations with several clinical cardiovascular research teams at the University of North Carolina. We will continue to correlate genetic variations within the GARF3 gene with GRAF3 expression and a number of cardiovascular end-points including vascular stiffness and intermediate disease outcomes. The completion of these aims will add significantly to our understanding of the development and pathophysiologic consequences of hypertension and to novel and perhaps individualized approaches to the treatment of this prevalent and debilitating disease.